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problem in selecting clones! - (Aug/04/2009 )

I am trying to generate a stable clone in various ca. cell lines like HCT116, MCF7, BT549, ASPC1, PANC1 etc. wherein I have to use Neomycin for selecting the clones also there is EGFP in it. The problem is that I have a mixed population of cells (GFP expressing and non-GFP) even after 7-10 passages after transfection which I am not able to kill using neomycin at 500ug/ml. In some flasks I put 1000ug/ml Neo, but did'nt helped. What should I do? Please give your suggestions!
Thanks in advance.


You might need to go for single cell selection or isolate a gfp expressing colony via cloning rings. i guess your cells might have been succesfully selected but some of them lost the gfp expression. these you won't get rid of by increasing the g418 concentration. I'd isolate single cells or gfp positive colonies and grow them up.